Alzheimer disease (AD) is characterized by neuronal loss, especially in the cortex and hippocampus, accompanied by accumulation in the brain of extracellular neuritic plaques containing -amyloid (A) and of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. AD patients also present with other features that have received less attention, including aberrant cholesterol, phospholipid, and calcium homeostasis, and altered mitochondrial function and dynamics. Presenilin-1 (PS1), presenilin-2 (PS2), and ?-secretase activity, which processes the amyloid precursor protein (APP) to generate A, are all located predominantly in a specialized subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAM). MAM is involved in the regulation of cholesterol and phospholipid metabolism, calcium homeostasis, and in mitochondrial function and dynamics. Recently, we showed that MAM is lipid raft-like domain and that cells from AD patients have massively upregulated MAM activity and increased ER mitochondrial connectivity, resulting in altered cholesterol, phospholipid and calcium homeostasis, and aberrant mitochondrial dynamics, which may help explain many of the biochemical and morphological features of the disease. Based on these findings, we believe that MAM dysfunction and altered ER-mitochondrial connectivity are early causative events in the pathogenesis of AD. We now propose studies aimed at understanding MAM function from a basic science standpoint, with the ultimate goal of applying this knowledge translationally. Specifically, we will (1) analyze MAM function in cells and tissues that are more AD-relevant, including PS-deficient human neuroblastoma cells, human induced pluripotent stem cells differentiated into neurons, and tissues and neurons explanted from PS1 knock-in mice; (2) analyze presenilins and ?-secretase regulation in MAM versus other compartments, such as bulk ER or the plasma membrane, while also assessing the role of other components (e.g. APH-1, nicastrin, PEN2) and regulators (e.g. CD147, GSAP, and TMP21) of the ?-secretase complex; and (3) understand the role of ER-mitochondrial communication in the pathogenesis of AD, by tethering ER to mitochondria at various fixed distances, using novel ER-mitochondria crosslinking plasmids, in order to mimic ER-mitochondrial connectivity in a presenilin-independent manner.